This project aims to understand the density, intracellular processing, PDZ interactions, trafficking, and possible dimerization of the GABA transporter mGAT1, by studying knock-in mice carrying fusions between mGAT1 and fluorescent proteins (XFP, where G = green; C, cyan; Y, yellow). The major tool is quantitative fluorescence microscopy. A mGAT1-XFP knock-in mouse strain will be developed that preserves a crucial PDZ interaction, mGAT1 will be counted in the synaptic structures of mGAT1-XFP knock-in mice. GAT1 interactions will be studied using hippocampal and cerebellar culture systems for the mGAT1-XFP mice. mGAT1-XFP mice will be mated with knockout mice for several synaptic proteins known to interact with GAT1, in order to study the effects on GAT1 distribution, trafficking, and sorting, using the culture system. The hypothesis that GAT1 dimerizes in vivo will be tested using fluorescent resonance energy transfer (FRET) between CFP-mGAT1 and YFP-mGATI. The hypothesis will be tested that GAT1 is carried on a special vesicle, and the number of mGAT1 molecules in a vesicle will be counted using evanescent wave- total internal reflection fluorescence (TIR-FM) microscopy. Also the project aims to understand the functions of GAT1 by continuing to study the intron 14-neo-mGAT1 strain, which is essentially a GAT1 knockout. The major tools are whole-animal physiology and electrophysiology. The motor phenotype produced in WT mice by acute administration of GAT1 inhibitors will be studied, to ask whether acute blockade explains the motor and behavioral anomalies of the intron 14-neo-mGAT1 strain. GAT1 will be selectively reintroduced in various brain areas and cell types to study the motor defects, by mating with various strains that have restricted expression of cre recombinase. Electrophysiology of the knockout will be studied. Similar knowledge will also be gathered about another GABA transporter, GAT3, by replicating the research program for GAT3-XFP mice. A GAT3-XFP fusion protein will be selected that displays proper function, sorting, and targeting. Knock-in mice will be constructed bearing this protein. Density, intracellular processing, trafficking, and possible dimerization of GAT3 will be studied. Similar knowledge will be sought about the serotonin transporter, SERT, by replicating Specific Aim 1 for SERT-XFP knock-in mice. Neurotransmitter transporters are targets for many modern drugs of therapy and abuse.